Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Abstract

Human immunodeficiency virus type 1 (HIV‐1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2′,3′‐dideoxy‐3′‐thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild‐type and 3TC‐resistant HIV‐1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild‐type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC‐resistant RT mutants produce significantly shorter cDNA molecules than the wild‐type enzyme [Met (wt)>Val>Ile], indicating that 3TC‐resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC‐resistant HIV‐1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC‐treated patients that develop drug‐resistant HIV‐1 variants, this may be of considerable clinical benefit.

YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study byin vitrofull-length viral DNA transfection

“…[11][12][13] Although lamivudine has a strong antiviral effect, the development of a resistant strain of virus presented a serious problem. 32 Lamivudine-resistant HIV was revealed to have amino acid substitutions in the YMDD motif of reverse transcriptase, 20,[33][34][35] and recently these substitutions have also been reported in HBV. [14][15][16][17] In addition, other mutations (phenylalanine [F]-to-L at amino acid 501 or an L-to-M substitution at amino acid 515) have been described in the B-domain of reverse transcriptase 18 of HBV during lamivudine and famciclovir treatment.…”

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

“…Virus production was determined by CA-p24 enzyme-linked immunosorbent assay. 25 Virus titers were determined by serial dilution of virus on SupT1 cells that were transduced for 6 h. Doxycycline (1000 ng ml À1 ) was added to the cells for 72 h, cells were subsequently harvested and GFP expression was measured using flow cytometry.…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…The best evidence for an impact of cell type on fitness has been obtained for HIV-1 mutants that are resistant to nucleoside analog inhibitors of the viral reverse transcriptase. For example, the replication fitness of M184V, which is resistant to lamivudine, was reduced in primary human PBMCs but was indistinguishable from that of wild-type HIV-1 in a lymphoid cell line (10). Purified reverse transcriptase with the M184V mutation showed reduced processivity of polymerization that was accentuated at low nucleotide concentrations (10).…”

“…For example, the replication fitness of M184V, which is resistant to lamivudine, was reduced in primary human PBMCs but was indistinguishable from that of wild-type HIV-1 in a lymphoid cell line (10). Purified reverse transcriptase with the M184V mutation showed reduced processivity of polymerization that was accentuated at low nucleotide concentrations (10). Since PBMCs are known to have lower concentrations of nucleotides than T-cell lines, this finding offers a potential biochemical mechanism for the differences in fitness of this mutant in primary cells versus cell lines.…”

“…M184V occurs frequently in viral isolates from patients failing lamivudine or emtricitabine therapy (63,153). M184V has a fitness similar to that of the wild type in most T-cell lines where nucleotide concentrations are high (10) but reduced fitness in primary cells that have limited nucleotide pools, such as PBMCs and macrophages (3,10,129). Of note is that there are some more-recent studies that have found reduced fitness of this mutant in cell lines (35,48).…”